Characterization of genes encoding diterpene cyclases responsible for phytoalexin biosynthesis in rice.

从水稻中分离出14种二萜类植物抗毒素：momilactones，phytocassanes和oryzalexins。我们已经分离了编码负责这些植物抗毒素的生物合成的二萜环化酶的cDNA; OsCPS 2、OsCPS 4、OsKSL 4、OsKSL 7、OsKSL 8、OsKSL 10。该项目是为了表征环化酶基因。1)基因表达模式：0 sCPS 2、0 sCPS 4、0 sKSL 4、0 sKSL 7和0 sKSL 8在根中的表达水平高于在地上部中的表达水平。0 sCPS 2、0 sCPS 4、0 sKSL 4和0 sKSL 7在水稻果实中的表达水平从抽穗期到开花期急剧增加。利用绿色荧光蛋白的瞬时测定表明，这些二萜环化酶运输薄质体。2)同源基因的分离：从水稻KS样同源基因cDNA中分离到OsKSL 5和OsKSL 6，分别编码对映庚二烯-8，15-二烯合酶和对映贝壳杉-15-烯合酶。OsCPS 2和OsCPS 4的两个同源cDNA分别从小麦和禾本科其他物种中分离得到。3)水稻二萜类植物抗毒素与抗病性的关系：受稻瘟病菌侵染后，6个基因的表达量增加。我们没有成功地筛选Tos 17插入敲除突变体和通过RNAi产生敲低突变体。4)OsCPS 2与负责赤霉素生物合成的OsCPS 1的比较：OsCPS 2的异位表达挽救了OsCPS 1缺失突变体的严重矮化表型。该结果表明OsCPS 1的表达调控与OsCPS 2的表达调控不同。OsCPS 2与OsCPS 1的基本酶性质基本相同，如二价阳离子需求量、最适pH、最适温度和动力学参数。然而，OsCPS 2的活性被底物香叶基香叶基二磷酸和赤霉素生物合成抑制剂AMO 1618抑制的程度小于OsCPS 1。

From rice 14 diterpenoid phytoalexins were isolated; momilactones, phytocassanes, and oryzalexins. We have isolated cDNAs encoding diterpene cyclases responsible for biosynthesis of these phytoalexins; OsCPS2, OsCPS4, OsKSL4, OsKSL7, OsKSL8, OsKSL10. This project was for characterization of the cyclase genes. 1) Gene expression patterns: The expression levels in roots of OsCPS2, OsCPS4, OsKSL4 OsKSL7, and OsKSL8, were higher than those in shoots. The expression levels of OsCPS2, OsCPS4, OsKSL4, and OsKSL7 were drastically increased in the rice fruits from heading stage to flowering stage. Transient assay using green fluorescence protein suggested that these diterpene cyclases are transported thin plastids. 2) Isolation of their homologues: From rice KS like homologue cDNAs, OsKSL5 and OsKSL6, which encode ent-pimara-8,15-diene synthase and ent-kaur-15-ene synthase respectively, were isolated. Two homologue cDNAs of OsCPS2 and OsCPS4 were isolated from wheat, other species in Poaceae. 3) Relationship of rice diterpene phytoalexins with pathogenic tolerance: The expression levels of six genes increased after infection by Magnaporthe grisea. We did not succeed in screening Tos 17-insertion-knock-out mutants and production of knock-down mutants by RNAi. 4) Comparison of OsCPS2 to OsCPS1 that is responsible for gibberellin biosynthesis: The severe dwarf phenotype of mutants lacking in OsCPS1 were rescued by ectopic expression of OsCPS2. This result indicated that regulation of OsCPS1 expression is different from that of OsCPS2 expression. The basic enzymatic properties of OsCPS2 were almost same as those of OsCPS1, such as divalent cation requirement, optimal pH, optimal temperature and kinetic parameter. However, the activity of OsCPS2 was suppressed by both of substrate geranylgeranyl diphosphate and a gibberellin-biosynthesis-inhibitor AMO 1618 less than that of OsCPS1.